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«(p)ppGpp and its role in bacterial persistence: New challenges» 1

Olga Pacios1,6, Lucia Blasco1,6 Inés Bleriot1,6, Laura Fernandez-Garcia1,6, Antón 2

Ambroa1,6, María López1,2,6, German Bou1,6,7, Rafael Cantón3,6,7, Rodolfo Garcia-3

Contreras4, Thomas K. Wood5 and Maria Tomás1,6,7* 4

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1. Microbiology Department-Research Institute Biomedical A Coruña (INIBIC), Hospital 7

A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain. 8 2. National Infection Service Laboratories, Public Health England, Colindale, UK. 9 3 Servicio de Microbiología, Hospital Ramón y Cajal and Instituto Ramón y Cajal de 10

Investigación Sanitaria (IRYCIS), Madrid, Spain. 11 4.Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad 12

Nacional Autónoma de México, Mexico City, Mexico. 13 5.Department of Chemical Engineering, Pennsylvania State University, University Park, 14

PA, United States. 15 6. Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) 16

of Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC). 17 7. Spanish Network for the Research in Infectious Diseases (REIPI).

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* Correspondence: ma.del.mar.tomas.carmona@sergas.es (MT); Tel: +34-981-176-23

399; Fax: +34-981-178-273 24

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Keywords: persistence, (p)ppGpp, TA systems, slow growth, PRDP. 33

Running title: (p)ppGpp and bacterial persistence 34

AAC Accepted Manuscript Posted Online 27 July 2020Antimicrob. Agents Chemother. doi:10.1128/AAC.01283-20Copyright © 2020 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 35

Antibiotic failure is not only due to the development of resistance by pathogens, but it can also 36

often be explained by persistence and tolerance. Persistence and tolerance can be included in 37

the “persistent phenotype”, with high relevance for clinics. Two of the most important 38

molecular mechanisms involved in tolerance and persistence are toxin-antitoxin (TA) modules 39

and signaling via guanosine pentaphosphate/tetraphosphate (p)ppGpp, also known as “magic 40

spot”). (p)ppGpp is a very important stress alarmone which orchestrates the stringent 41

response in bacteria; hence, (p)ppGpp is produced during amino acid or fatty acid starvation 42

by proteins belonging to the RelA/SpoT homologs family (RSH). However, (p)ppGpp levels can 43

also accumulate in response to a wide range of signals, including oxygen variation, pH 44

downshift, osmotic shock, temperature shift or even exposure to darkness. Furthermore, the 45

stringent response is not only involved in responses to environmental stresses (starvation for 46

carbon sources, fatty acids, phosphate or heat shock), but it is also used in bacterial 47

pathogenesis, host invasion, antibiotic tolerance and persistence. Given the exhaustive and 48

contradictory literature surrounding the role of (p)ppGpp in bacterial persistence, and with the 49

aim of summarizing what is known so far about the “magic spot” in this bacterial stage, this 50

review provides new insights into the link between the stringent response and persistence. 51

Moreover, we review some of the innovative treatments that have (p)ppGpp as a target, which 52

are in the spotlight of the scientific community as candidates for effective anti-persistence 53

agents. 54

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1. Introduction 55

Antimicrobial resistance crisis is a serious health problem worldwide. During the past fifty 56

years, very few new anti-infective molecules have been discovered (1). Hence, microbial 57

pathogens have been able to accumulate molecular mechanisms enabling them to counteract 58

antibiotics. 59

Nonetheless, there are more antibiotic evasion strategies other than resistance that are of 60

great interest, such as persistence and tolerance. Persisters are a subpopulation of cells that 61

are non-growing, non-replicative, dormant bacteria that exhibit transient high levels of 62

tolerance to antibiotics without affecting their MICs (2-4). Once the drug pressure is removed, 63

these persisters can rapidly regrow, thus returning to an antibiotic sensitive state. Moreover, 64

the persistent state can be maintained for hours up to days before persisters revert to an 65

antibiotic-sensitive cell type, resuming growth under drug-free conditions (5). 66

The term “triggered persistence” has been recently coined to indicate a form of persistence 67

that is induced by particular signals, such as starvation and nutrient transitions, acid- and 68

oxidant-stress, DNA damage, subinhibitory concentrations of antibiotics and intracellular 69

infections (6). 70

Similar to persisters, tolerant cells are populations of bacteria that can also overcome 71

antibiotic therapy. Tolerance allows cells to temporarily counteract the lethal consequences of 72

high doses of antibiotics, maintaining their vital processes slowed (4, 7, 8). Also, tolerant 73

bacteria arise when the whole population slows its growth (e.g., stationary-phase) whereas 74

persister bacteria are a small subpopulation of the population (4). 75

Both tolerant and persistent bacteria can be included in the “persistent phenotype”, which has 76

high relevance in clinics because: (i) there is evidence that persistent cells are responsible for 77

relapses of infections, which is common in tuberculosis, cystic fibrosis and Lyme disease (9, 78

10); (ii) antibiotic therapy does not effectively work against these types of infections; (iii) 79

persisters are responsible for the majority of biofilm-associated infections (11, 12) and (iv) they 80

are associated with better survival of bacteria inside macrophages (13). Furthermore, persister 81

cells can also survive in immune-compromised patients and in patients in whom antibiotics do 82

not effectively kill pathogenic bacteria, as they might deploy immune-evasion strategies (14). 83

Differences between resistance, persistence and tolerance have been established; 84

nonetheless, there are also relationships among these bacterial populations which are worthy 85

of consideration. Despite evidence showing that tolerance and persistence to antibiotics 86

promote the evolution of resistance in bacteria (8, 14, 15) both mechanisms are currently 87

underestimated by the scientific and medical communities. 88

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There are several molecular mechanisms involved in bacterial persistence and tolerance to 89

antibiotics, reviewed by Trastoy and colleagues (2018), which include: (p)ppGpp network; 90

toxin-antitoxin (TA) system; the quorum sensing (QS) system; drug efflux pumps; reactive 91

oxygen species (ROS); the SOS response; and RpoS (sigma factor of stationary phase) (7). 92

(p)ppGpp orchestrates the stringent response (SR) in bacteria, thus it is produced during 93

nutrient stress (such as amino acid or fatty acid starvation) by proteins belonging to the 94

RelA/SpoT Homologs family (RSH) (16). In E. coli, RelA is the (p)ppGpp synthetase I or GTP-95

pyrophosphokinase that synthesizes (p)ppGpp from GTP/GDP and ATP, whereas SpoT is a 96

bifunctional (p)ppGpp synthetase II or pyrophosphohydrolase (17). However, (p)ppGpp levels 97

in bacteria do not depend exclusively on nutrient availability (18), since it can also accumulate 98

in response to a wide range of signals, including oxygen variation (19), pH downshift (20), 99

osmotic shock, temperature shift (21) or even exposure to darkness (22). Furthermore, the SR 100

is not only involved in responses to environmental stresses (starvation for carbon sources, 101

fatty acids, phosphate or heat shock), but also in bacterial pathogenesis (23), host invasion 102

(24), antibiotic tolerance and persister cell formation (25). 103

Given the exhaustive and contradictory literature surrounding the role of (p)ppGpp in bacterial 104

persistence, and with the aim of summarizing what is known so far about the “magic spot” in 105

this bacterial stage, this review provides new insights into the link between the SR and 106

persisters. Finally, we have reviewed and discussed some of the innovative treatments that 107

have (p)ppGpp as a target, which are in the spotlight of the scientific community as candidates 108

of effective anti-persistence agents. 109

2. (p)ppGpp as a key regulator 110

It was in 1969 that Cashel and Gallant described for the first time guanosine 111

penta/tetraphosphate (26). During these fifty years, many functions have been attributed to 112

this alarmone as it plays a key role in the physiology of bacteria, mainly controlling energetic 113

metabolism (26, 27) but also their virulence and immune evasion (26). Despite being widely 114

studied in the model organism E. coli, (p)ppGpp behaves differently in other species and its 115

regulation changes among phylogenetically-related bacterial groups (27). In E. coli, the 116

accumulation of (p)ppGpp causes the differential expression of approximately 500 genes, as it 117

activates RpoS and RpoE (the stress response sigma factor for misfolded proteins in the 118

periplasm) (28). Also in E. coli, (p)ppGpp directly inhibits DNA primase (29), and is thought to 119

inhibit the synthesis of rRNA, which also affects translation globally, by regulating the 120

transcription of the ribosomal modulation factor (Rmf) (30). More specifically, in response to 121

stresses such as amino acid starvation, in Gram negative bacteria (p)ppGpp binds to the RNA 122

polymerase inducing an allosteric signal to the catalytic Mg2+ site, which severally decreases 123

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transcription causing a global re-wiring of the gene expression profile. Taken together, all 124

these changes lead to dormancy or slow growth for most cells (Figure 1) (31, 32). 125

Most Gram-positive bacteria possess only one long RSH, named Rel, which has both (p)ppGpp 126

synthetase and hydrolase activities (33, 34) together with a short RSH, named SAS (Small 127

Alarmone Synthetase) or SAH (Small Alarmone Hydrolase) (35). Nonetheless, the existence of a 128

diverse population of (p)ppGpp synthetases and hydrolases in bacteria has been demonstrated 129

(35-38). 130

There is evidence that (p)ppGpp is related to antibiotic tolerance and persister cell formation 131

(25, 39-42). For example, increased levels of (p)ppGpp inhibit negative supercoiling of DNA in 132

E. coli, thus preventing DNA replication and transcription, resulting in tolerance towards 133

ofloxacin and quinolones (Figure 1) (43, 44). Persister cells tend to form in biofilms, in which 134

the bacterial cells are embedded in a three-dimensional matrix that provides protection during 135

pathogenesis and other conditions. Thus, it is logical that bacteria in biofilms encounter limited 136

access to nutrients and therefore display the SR (45). Consistently, different groups have 137

shown that multidrug tolerance of P. aeruginosa and E. coli grown in biofilms depends on 138

(p)ppGpp (45-47). Also, in 2014, Helaine and colleagues performed an experiment where they 139

studied the invasion of mouse macrophages by Salmonella enterica, and they observed that 140

(p)ppGpp production by bacteria residing in acidified vacuoles of macrophages was required 141

for persistence (13). 142

3. (p)ppGpp as a mediator in bacterial growth. 143

Accumulation of (p)ppGpp promotes transcriptional alterations in the bacterial cell, such as 144

general repression of rapid growth genes and activation of genes involved in amino acid 145

biosynthesis and survival to stress (29). Therefore, loss of (p)ppGpp in some conditions (for 146

example minimal medium) leads to amino acid auxotrophy of the whole population and an 147

increased survival due to high tolerance. Similarly, many other conditions or mutations that 148

decrease bacterial growth rate have been shown to induce the same tolerant phenotype (48). 149

Several authors suggested that, at least in Salmonella, there was no specific molecular 150

pathway underlying bacterial persistence, but that slow growth was the main factor to induce 151

persistence (49, 50). Regarding the involvement of (p)ppGpp in bacterial growth, it has been 152

shown that relA spoT double null mutants of E. coli, completely depleted of (p)ppGpp, are 153

more elongated than wild-type cells (51). LpxC, a key enzyme that catalyzes one of the first 154

steps in the synthesis of lipopolysaccharide (LPS) in E. coli, is degraded during slow growth but 155

stabilized when cells grow faster (Figure 1) (52). Hence, in 2013 it was reported that in relA 156

spot double null mutants, there was a deregulation of LpxC degradation, resulting in rapid 157

proteolysis in fast-growing cells and stabilization during slow growth, in opposition to the 158

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normal state where (p)ppGpp is present (52). In 2015, Yamaguchi and colleagues found that 159

elevated levels of (p)ppGpp led to inhibition of bacterial growth by interfering with the FtsZ 160

protein assembly in Salmonella paratyphi A (53). FtsZ is a protein essential for the prokaryotic 161

cell division that needs to form linear structures and has a GTP binding site; however, 162

increased levels of (p)ppGpp (20-fold higher than the required GTP levels) causes FtsZ to form 163

helical structures unable to form the Z-ring at the division site (53), which impairs the division 164

of the bacterial cell and, therefore, the population growth. In short, high levels of (p)ppGpp 165

can promote persistence by halting growth in a subpopulation (even in a rich-nutrient 166

medium), while absence of this alarmone can contribute to tolerance (by preventing the whole 167

population to appropriately handle a nutritional stress). 168

4. Association between other molecular mechanisms and (p)ppGpp in persistence 169

According to Trastoy and colleagues (7), some of the molecular mechanisms that have been 170

related to bacterial persistence are TA systems, QS and secretion systems, efflux pumps, SOS 171

system and ROS response (Figure 2A). We focus only on those where a link between (p)ppGpp 172

or SR and persistence has been reported: TA systems, efflux pumps and ROS systems (Figure 173

2B): 174

4.1 TA systems 175

The persistent state actually describes many different growth-arrested physiologies and it has 176

been associated, during years, with the activity of TA systems, at least partially (54, 55). TA 177

systems are a module of two genes encoding a stable toxin and a usually unstable antitoxin 178

which is degraded under stress conditions; however, bound antitoxin to toxin is not likely the 179

source of free toxin since the two proteins are bound tightly (56) (Figure 2A and B). Once 180

unbound toxin is produced, the harmful toxin slows growth, maintains plasmids (57), inhibits 181

phage (58) and induces biofilm formation (59, 60). TA systems are widely distributed in 182

pathogenic bacteria and found in the bacterial chromosome, plasmids and bacteriophages (61, 183

62). 184

In 1983, Moyed and Bertrand identified the first persistence gene related to increased survival 185

in presence of ampicillin in E. coli (63). They discovered a high persistence, gain of function 186

mutation, named hipA7; hipA encodes the toxin of the HipA/HipB TA system. Similarly, the 187

second link between TA systems and persistence was reported by Aizenman and colleagues in 188

1996, when they observed that (p)ppGpp was required to activate MazF toxicity, the toxin of 189

the MazF/MazE TA system (64). In 2003, Korch and colleagues studied the role of HipAB TA 190

system in persistence, showing that the ability of E. coli to survive to a prolonged exposure to 191

penicillin was due to two mutations in the non-toxic hipA7 allele (65). Both mutations, G22S 192

and D291A, were required for the full range of phenotypes associated with high persistence, 193

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increasing 1,000- to 10,000-fold the frequency of persisters. Moreover, in this study they also 194

demonstrated that relA knockouts diminished the high persistent phenotype in hipA7 mutants, 195

and that relA spoT knockouts completely eliminated this high persistence, suggesting that 196

hipA7 facilitates the establishment of the persistent state by inducing (p)ppGpp synthesis (65). 197

This result was confirmed in 2011 by Nguyen and colleagues in P. aeruginosa, where a relA 198

spot double mutant led to a decrease of 68-fold in persistence (45). Interestingly, Correia and 199

colleagues demonstrated that HipA exhibits a eukaryotic serine-threonine kinase activity, 200

required for both the inhibition of cell growth and the stimulation of persister cells (66). In 201

2009, Schumacher and colleagues suggested that HipA inhibited cell growth by the 202

phosphorylation of the essential Elongation Factor Tu (EF-Tu), involved in translation (67). 203

Notwithstanding, in 2013, Germain and colleagues challenged the previous model in which 204

HipA inactivated translation by the phosphorylation of the EF-Tu, and proposed a novel 205

paradigm in which HipA inactivates the glutamyl-tRNA-synthetase (GltX) by phosphorylation of 206

the conserved Ser239 near the active center (68). In addition, the authors claimed that this 207

phosphorylation inhibited aminoacylation, which halts translation and induces the SR by the 208

generation of “hungry” codons at the ribosomal A site. Thus, RelA binds to the ribosome, is 209

activated, and increases (p)ppGpp levels that inhibit translation, transcription, replication and 210

cell-wall synthesis, thereby leading to slow growth, multidrug tolerance, and persistence 211

(Figure 1) (47, 68-70). 212

In this context, further studies have been conducted to unravel the association of TA systems 213

and (p)ppGpp in persistence. In 2012, Gerdes and Maisonneuve reported that the removal of 214

10 mRNAse-encoding TA loci of E. coli led to a dramatic decrease of persistence in the 215

presence of the antibiotic (71). A similar phenomenon was observed with a mutant lacking Lon 216

protease, which indicated that TA systems and Lon protease were somehow correlated and 217

both implicated in the persistence of E. coli (72). Since many antitoxins of E. coli were 218

degraded by Lon protease, Gerdes and Maisonneuve hypothesized that HipB was one of the 219

targets. In E. coli, Lon can be activated by polyphosphate (PolyP), synthesized by 220

polyphosphate kinase (PPK) and degraded by an exopolyphosphatase (PPX). PPX is inhibited by 221

(p)ppGpp, which leads to an increase in PolyP. Hence, these authors claimed that high levels of 222

(p)ppGpp associated with persistence inhibited PPX, thus allowing PolyP to activate Lon 223

protease, which would degrade the HipB antitoxin. Furthermore, they suggested that PolyP 224

functions as an intracellular signaling molecule controlled by the SR, and (p)ppGpp reprograms 225

the cells to survive starvation. (71). In 2014, these authors completed this model by adding one 226

additional step, which is a speculative positive-feedback loop that ensures even more synthesis 227

of (p)ppGpp (42). Thus, the model predicts that the degradation of HipB enables free HipA to 228

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phosphorylate GltX and inhibits charging of tRNA-glu. When uncharged tRNAs enter the 229

ribosomal A site, RelA-dependent synthesis of (p)ppGpp is triggered (Table 1 and Figure 1) 230

(42). Even though this model became very influential, the authors retracted the key articles in 231

2018, claiming that the apparent inhibition of persistence in the multiple-deletion strain was 232

due to an inadvertent lysogenisation with the bacteriophage Φ80, a contaminant that caused 233

artefacts in their experiments (73). In addition, there were conflicting reports for the 234

(p)ppGpp/PolyP/Lon persistence model, such as that Lon protease is not activated but 235

deactivated by PolP (74), or not required for persistence (Table 1 and Figure 2B) (75). 236

In 2019, Pontes and Groisman studied the implication of TA modules and (p)ppGpp in the 237

persistence of Salmonella and they revealed that low cytoplasmic Mg2+ induced tolerance to 238

antibiotics independently of (p)ppGpp and TA modules (Table 1) (49). In fact, a relA spoT 239

double mutant of Salmonella, unable to produce (p)ppGpp, exhibited similar tolerance to 240

antibiotics after growing in low Mg2+ than the wild-type strain. The same phenomenon 241

occurred with the mutant strain lacking 12 TA systems (Δ12TA). However, when the antibiotic 242

treatment was added at neutral pH, they saw five- to eightfold fewer persisters compared to 243

WT both in the Δ12TA strain and in double mutant relA spoT (49). Nonetheless, when they 244

deleted one single TA module found that this mutation had no effect in persistence (49) 245

(Figure 2B). 246

Recently, Song and Wood (2020), claimed that TA systems were not involved in the formation 247

of persistent bacteria (76). The many contradictory observations in diverse experimental 248

setups lead to no clear conclusive understanding of the implications of TA systems in 249

persistence and further work is needed. 250

4.2 Efflux Pumps 251

Efflux pumps are proteic complexes that allow bacteria to draw out intracellular toxins or 252

antibiotic molecules. Some genes encoding efflux pumps are upregulated in cells that 253

constitute biofilms, which are composed mainly by non-growing, persistent cells (77). This 254

upregulation in persister cells can be triggered by different signals, such as ROS response, QS 255

and (p)ppGpp (78). 256

The first data that linked SR with efflux pumps were apported by Wang and colleagues, who 257

observed that a ppx2 (encoding the exopolyphosphatase that degrades poly(P)) knockout 258

mutant of M. tuberculosis (where poly(P) and (p)ppGpp accumulate) exhibited increased levels 259

of several efflux genes, including iniA, iniB, mmpL10 and Rv2459 (Figure 2B). Notwithstanding, 260

the authors concluded that the element which contributed the most to isoniazid tolerance in 261

this mutant was the changes in the cell wall thickness, as they limited the diffusion of polar 262

molecules such as isoniazid (79). 263

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Some years later, in 2018, Ge and colleagues described that a glucose/galactose transporter of 264

H. pylori, Hp1174, functions as an efflux pump and is highly expressed in biofilm-forming and 265

MDR H. pylori strains. This transporter, encoded by the gluP gene, is upregulated by SpoT (78). 266

A H. pylori mutant lacking gluP gene and its product Hp1174 constituted an unstructured 267

biofilm whose matrix was damaged. As this study revealed that SpoT enzyme upregulates 268

Hp1174 in persistent biofilm-forming cells, and provided that the transcription of this gene is 269

controlled by an alternative sigma factor (σ54), they hypothesized that SpoT may upregulate 270

the expression of gluP by a σ54-dependent transcription. 271

Finally, Pu and colleagues demonstrated that β-lactam-induced E. coli persisters exhibited less 272

cytoplasmic drug accumulation because of an enhanced efflux activity compared to non-273

persister cells. Combining time-lapse imaging and mutagenesis techniques, scientists 274

determined a positive correlation between tolC expression and arising of E. coli persisters (80). 275

4.3 ROS response 276

The reactive oxygen species (ROS) are produced as a natural response to the normal 277

metabolism of the oxygen and perform important functions in cell signaling and homeostasis. 278

However, when cells are exposed to environmental pressure, such as antibiotics, UV, or heat 279

pressure, ROS levels can increase; this increase can cause damages in the DNA, lipids and 280

proteins, which subsequently leads to cell death. Like all molecular mechanisms, ROS are 281

subject to regulation (Figure 2B). Among the molecules capable of eliminating ROS, we find 282

enzymes, such as superoxide dismutase (SOD) and catalase, as well as antioxidant agents, such 283

as glutathione and vitamin C. However, when an increase in ROS levels occurs due to an 284

imbalance between the production and elimination mechanisms, cells are said to be subject to 285

oxidative stress (7). 286

The relationship between ROS response and persistent bacteria are widely described in the 287

literature. Nguyen and colleagues, in 2011, showed that the survival of multidrug-tolerant 288

persisters in biofilms of P. aeruginosa was largely dependent on catalase or SOD enzymes, 289

which are under the control of the (p)ppGpp signaling (45). Along the same lines, the study of 290

Khakimova and colleagues (2013) demonstrated that the SR regulates catalases, likely through 291

a complex interplay of regulators (81) (Figure 2A and B). Furthermore, they also demonstrated 292

that H2O2 and antibiotic tolerance were the result of a balance between prooxidant stress and 293

antioxidant stress (81). Similarly, the work of Molina-Quiroz and colleagues (2018), gave more 294

evidence of the relationship between oxidative stress and bacterial survival to antibiotics. In 295

this study, the authors demonstrated the impact of ROS on the generation of persister cells, 296

exposing the cultures of a WT strain and its corresponding mutant lacking the cAMP synthase 297

adenylate cyclase (ΔcyaA) under the antibiotic pressure of ampicillin in the presence and 298

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absence of oxygen. For both strains, they observed a 100-fold increase of ampicillin survival in 299

absence of oxygen compared to the strain under aerobic conditions. This study concludes that 300

the damage that ROS cause in the DNA was regulated by cAMP, a negative regulator of 301

persistence in uropathogenic E. coli (82). 302

5. PRDP: (p)ppGpp ribosome dimerization persister model 303

Song and Wood, proposed a novel model in which the alarmone (p)ppGpp would generate 304

persister cells by inactivating ribosomes via the Rmf and the hibernation promoting factor 305

(Hpf) (Figure 1 and 2B, Table 1) (83). Among their findings, the following should be highlighted: 306

(i) E. coli persisters contain a large fraction of inactivated 100S ribosomes; (ii) Rmf and Hpf 307

induced persistence, and the inactivation of these proteins increased the single cell persister 308

resuscitation, and (iii) (p)ppGpp did not affect the single-cell persister resuscitation. In another 309

work it was reported that (p)ppGpp induced the Hpf, converting the 90S ribosomes into 100S 310

ribosomes, and that overproduction of Rmf and Hpf increased persistence as well as reduced 311

single cell resuscitation (84). Furthermore, authors based their theory on the fact that 312

(p)ppGpp inhibits the ribosome-associated GTPase Era, essential in the assembling of 313

ribosomal 30S subunits in Staphylococcus aureus (85). Hence, a connection between (p)ppGpp 314

and persistence via ribosome dimerization was demonstrated. 315

In 2019, Libby and colleagues showed that there was an enormous variability in sasA 316

expression (the gene encoding SasA in B. subtilis) among bacterial cells, linking a higher 317

expression of sasA with an increase in antibiotic survival (86). (p)ppGpp synthetases in B. 318

subtilis, such as SasA, are important in ribosome dimerization, as YwaC induces the 319

transcription of yvyD gene, whose product, YvyD protein, is essential for the dimerization of 320

70S ribosomes (87). 70S dimers are similar to the above-mentioned 100S ribosomes in E. coli, 321

therefore translationally inactive. These results agree with the PRDP model, where (p)ppGpp 322

would induce bacterial persistence by promoting ribosome dimerization and compromising the 323

translation inside the cells (83). 324

6. Anti-persisters treatments with (p)ppGpp as a target 325

Most antibiotics used in clinics target active metabolic processes. Therefore, bacteria that 326

exhibit a reduction in metabolism and growth rate, such as tolerant or persistent cells, are not 327

a target for the classic antibiotics. In the literature, a few reviews summarizing some of the 328

most useful strategies to combat persistent infectious diseases can be found (2, 88, 89). 329

As the activation of the SR leads to the shutdown of nearly all metabolic processes and the 330

entrance into a state of dormancy, an interesting therapeutic approach to combat persistent 331

infections is the inhibition of the SR network. Hobbs and Boranson (2019) reviewed the recent 332

attempts that have been made to design and discover inhibitors of the SR, and they concluded 333

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that there are currently two approaches: (i) inhibition of (p)ppGpp synthetases by using 334

(p)ppGpp analogs and (ii) inhibition of (p)ppGpp accumulation by using protein inhibitors (90). 335

i. Inhibition of (p)ppGpp synthetases by using (p)ppGpp analogs 336

Several in vitro studies using double relA spoT null mutants in E. coli have shown that this 337

bacterium lacks the (p)ppGpp and therefore has significantly reduced persistence to 338

antibiotics. In this context, some compounds that inhibit the (p)ppGpp production (thus SR), 339

had been developed to abolish persistence. Relacin, one of these compounds, was first 340

designed in 2012 by Wexselblatt and colleagues, and was shown to inhibit Rel-mediated 341

(p)ppGpp synthesis, leading to the death of B. subtilis with an estimated IC50 of 200 μM (Table 342

2) (91). Importantly, relacin also prevented the formation of spores and biofilm in this species. 343

Before bacterial death, relacin also induced a prolonged exponential phase. In 2017, Syal and 344

colleagues performed a slight modification of relacin and found that cells of Mycobacterium 345

smegmatis treated with this molecule were not able to establish any biofilm and were 346

elongated, showing exactly the same phenotype as a rel- mutant (92). Interestingly, this 347

relacin-derived compound lacked toxicity with human red blood cells and has good 348

permeability into the human lung epithelial cells. One of the most persistent pathogens, M. 349

tuberculosis, could be potentially targeted with this (p)ppGpp inhibitor if its evaluation in 350

humans turns to be effective and safe (Table 2). 351

In order to find other inhibitors of Rel protein, Dutta and colleagues (2019) performed a high-352

throughput screening approach, using Rel from M. tuberculosis and a novel (p)ppGpp 353

synthetase assay, based on detection of AMP released after Rel catalyzes the transfer of 354

pyrophosphate groups from ATP to GTP/GDP (93). This screening led to the identification of 355

the most potent Rel inhibitor to date, the compound X9, which exhibited an IC50 of ∼15 μM 356

against purified Rel. At 4 μM, when M. tuberculosis was nutrient-starved, it enhanced its 357

susceptibility against isoniazid (Table 2). Even if the molecular mechanism by which X9 inhibits 358

Rel is not fully understood yet, this compound displays the most potent activity of any Rel 359

inhibitor to date. 360

ii. Inhibition of (p)ppGpp accumulation 361

A second approach to design anti-persistence strategies that target (p)ppGpp would be the 362

inhibition of the accumulation of this alarmone. Biofilms are very important in the 363

establishment and maintenance of many infections caused by pathogenic bacteria, therefore 364

some cationic peptides with anti-biofilm abilities have been tested and proposed to act via 365

disruption of the SR (11, 90). 366

The 1018 peptide (VRLIVAVRIWRR-NH2) is a small, synthetic, L-amino acid peptide derived 367

from a bovine host defense peptide. De la Fuente and colleagues (2014) first described that 368

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1018 marks (p)ppGpp for degradation, exhibiting potent activity against biofilms produced by 369

Gram-positive (S. aureus) and Gram-negative bacteria (E. coli, P. aeruginosa, K. pneumoniae or 370

A. baumannii), but not in planktonic cultures (94). They also observed that the 1018 peptide 371

prevented the biofilm formation and degraded the pre-formed biofilm (as old as 2 days). As an 372

overproduction of (p)ppGpp leads to resistance to the 1018 peptide, the authors suggested 373

that this peptide specifically targeted SR (Table 2). The same research group generated 374

another small peptide, called 1037, and observed its effects on biofilm formation and 375

swarming motility in P. aeruginosa, Burkholderia cenocepacia and Gram-positive Listeria 376

monocytogenes (Table 2) (95). They observed that 1037 reduced flagella-dependent swimming 377

in P. aeruginosa PA14, P. aeruginosa PAO1, and B. cenocepacia; consistently, transcriptomic 378

analysis revealed that, in the presence of 1037, several genes related to flagella were 379

downregulated by 2- to 3-fold. This is interesting because flagella are involved in biofilm 380

formation and swarming motility, both significantly inhibited by the action of 1037 in the 381

tested species (95). 382

Notwithstanding, the study of Andresen and colleagues in 2016 rejected the idea that 1018 383

peptide specifically targets the SR alarmone (p)ppGpp (96). Furthermore, they observed that in 384

P. aeruginosa this peptide showed anti-bacterial activity both in planktonic and in biofilm-385

derived cells. 386

Interestingly, Allison and colleagues published an innovative article in 2011 where they killed E. 387

coli and S. aureus persisters combining different metabolites with aminoglycosides (97). They 388

reported that glucose, pyruvate, mannitol or fructose significantly increased the PMF; this 389

leads to a higher uptake of the aminoglycoside and the consequent killing of the persisters, 390

either in vitro and in a mouse model carrying a catheter colonized by uropathogenic E. coli. In 391

conclusion, these findings mean that some of these PMF-stimulating metabolites might be a 392

good adjuvant to aminoglycoside to treat persistent, chronic infections (97). 393

7. Discussion 394

We have summarized the functions of (p)ppGpp regarding its role as a global transcription and 395

translation regulator of metabolism, slow growth and dormancy, nutrient starvation, different 396

kinds of stress, virulence, tolerance to antibiotics, persister cell formation and even persistence 397

inside macrophages. However, an accurate role of this alarmone in persistence has not been 398

determined yet. Clearly, evidence relates this molecule to the persistent phenotype, based on 399

its dominant role in the stress response of bacteria. 400

The diversity among the conclusions obtained by laboratories around the globe raised the 401

question of whether persisters in phylogenetically close organisms are produced through 402

different pathways (49, 98). Nevertheless, this seems unlikely as the SR is a universal, highly 403

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conserved network in many phyla, and all microbes use it to protect themselves against 404

different types of stress. 405

The relevance of the role of the ATP in the antibiotic persistence is revealed as tolerant cells 406

slow down their metabolism and persistent cells are quiescent. Pontes and Groisman (2019) 407

showed that Salmonella pre-exposed to chloramphenicol resisted the killing by bactericidal 408

antibiotics (49). However, contradictory results have been obtained from different groups 409

indicating that ATP does not control persistence (49, 99, 100), or even that persister cell 410

formation is based on reducing ATP (101-104). 411

The results of Pontes and Groisman (2019) (49), agree with the findings of both Hobby (105) 412

and Bigger (106) and with many other researchers who have shown that deliberate induction 413

of bacteriostasis promotes antibiotic tolerance (15). Whereas deliberate induction of 414

bacteriostasis overrides bacterial control of growth, it remains to be explored what 415

mechanisms promote growth arrest in individual cells. They showed that Salmonella persisters 416

emerged as a result of slow growth alone and transitory disturbances to core activities, 417

regardless of the underlying physiological process. They also performed studies with 418

Salmonella mutants lacking 12 TA modules and observed their implication in persistence in 419

some conditions (49). Kaldalu and colleagues (2019) claimed that there was no specific 420

molecular mechanism involved in persistence but this latter was simply produced by slow 421

growth of bacteria (50). 422

It remains unclear if efflux pumps and the SOS-system have a real link with (p)ppGpp. Despite 423

being important molecular mechanisms widely studied in pathogenic bacteria, there is still few 424

literature linking (p)ppGpp metabolism with these mechanisms, and it is even trickier the 425

association of these to persistence; a proof of that is the article of Ge and colleagues, where 426

they claim that bifunctional SpoT enzyme up-regulates the Hp1174 efflux pump of H. pylori, 427

contributing to biofilm formation (78). Regarding the role of ROS-system in persistence, 428

different research groups have demonstrated that (p)ppGpp and the SR regulate the 429

expression of antioxidant enzymes, e.g SOD or catalases, in order to avoid the intracellular 430

accumulation of ROS (82). Even if the intermediate regulators involved in this pathway need 431

further research, this opens the door to anti-persister therapies targeting SR (45, 81). 432

Traditionally, the persistence of the bacteria has been widely attributed to TA systems, as 433

Chowdhury and colleagues published in 2016, where they claimed that persister cells can form 434

in the absence of (p)ppGpp (although at much-reduced levels), mainly due to the effect of 435

production of any toxic protein (75). Nevertheless, Dr T. K. Wood, reported some years later an 436

essential role of (p)ppGpp in the establishment of persistence via induction of dimerization of 437

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ribosomes. In this new model, called PRDP and proposed by Song and colleagues in 2020, 438

there is evidence of a direct role of the magic spot in the persistent phenotype (83). 439

An interesting issue is the individual variability within a population of cells regarding their 440

tolerance to antibiotics. Whether this heterogeneity is regulated or, on the contrary, is an 441

unavoidable consequence of stochastic fluctuations, remains unknown. In 2004, Balaban and 442

colleagues showed that spontaneous persisters are rare, suggesting that they were not 443

stochastically produced (3). Similarly, the PRDP model also suggests that both persistence and 444

resuscitation are sophisticatedly-regulated by ribosome content and their activation status 445

(83). Finally, another hint of the regulation of SR was proposed by Libby and colleagues (2019), 446

based on the fact that SasA in B. subtilis has multiple sites of phosphorylation, which can 447

explain the cell-to-cell variability in sasA expression (86, 87). These differences may be the 448

reason for the physiologically relevant variability in (p)ppGpp levels and shed some light into 449

the heterogeneity within a bacterial population and their phenotypic variability. 450

There are some arguments in favor of the PRDP model: one is that Hpf, which converts 90S 451

ribosomes into inactivated 100S ribosomes in E. coli, is highly conserved in most bacteria (84); 452

secondly, other (p)ppGpp synthetases found in B. subtilis are essential for ribosome 453

dimerization in Gram-positive bacteria, generating translationally inactive ribosomes 454

associated with persistence (87). 455

In this review we have contrasted different models that have been proposed over many years 456

all of them aiming at answering the same question: what is the precise role of (p)ppGpp and SR 457

in bacterial persistence? Definitely, after comparing all those models we can conclude that 458

uncontrolled variables such as contaminants 459

(as Φ80 phage), particular setups that differ from lab to lab, artifacts that mislead to 460

conclusions, changes in the tested strains and, in short, different experimental conditions can 461

be some of the underlying reasons to explain the controversy around this question. 462

The current lack of effective antibiotics against multi-drug resistant, persister and tolerant 463

pathogens leads the urgency to develop new antibacterial treatments, as the anti-persistent 464

treatments targeting the (p)ppGpp network. The inhibitors of (p)ppGpp synthetases are good 465

candidates as antimicrobial agents, because of their high efficacy in avoiding biofilm formation 466

(91), in the loss of the persistent phenotype and even in the prolongation of exponential 467

phase, when bacteria replicate more actively, being more susceptible to antibiotics. This also 468

opens the door to the possibility of a combination of therapies (inhibitors of (p)ppGpp 469

synthetase with antibiotics) and to the establishment of potential synergies. Even if no effect 470

of the Rel inhibitor relacin was observed in E. coli, the authors suggested that this could be due 471

to the inability of relacin to penetrate Gram negative cells and reach its target. However, 472

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agents as polymyxins can destabilize the outer membrane in Gram negatives facilitating the 473

entrance of therapeutic molecules, e. g., relacin, or endolysins (108). The absence of known 474

(p)ppGpp synthetases in mammalian cells and the specificity of these inhibitors for the Rel 475

protein, make this protein a good candidate as an antibacterial agent. 476

The second strategy of inhibition of (p)ppGpp accumulation, supported by the 1018 and 1037 477

peptides, exhibited promising anti-persistent activities as they specifically targeted biofilm-478

forming cells and had no effect on planktonic cells (94, 95). According to the few studies that 479

focus on inhibiting the SR as an anti-persistent therapeutic approach, we can consider this as 480

an emerging field; therefore, further research and financial investment are needed to 481

efficiently prevent persistent, chronic and life-threatening infections. 482

8. Conclusion 483

The emergence of contradictory models about the involvement of the “magic spot” in bacterial 484

persistence highlights the need to deepen the studies in this field. In summary, one potential 485

strategy to fight persistent infectious disease resides in specifically targeting SR or (p)ppGpp of 486

pathogenic bacteria, but further knowledge is necessary to provide a better understanding of 487

the complexity of bacterial persistence, as well as its implications in clinics. 488

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Legends 508

Figure 1. Physiological pathways regulated by (p)ppGpp and the stringent response in E. coli. 509

Some of the most common human opportunistic pathogens are represented, such as 510

biofilm-forming P. aeruginosa, intestinal E. coli and skin-borne opportunistic pathogen 511

S. aureus. Focusing on E. coli: 1) increased levels of (p)ppGpp induce transcription of 512

RpoS, sigma factor for the stationary phase, and RpoE, the sigma factor that regulates 513

the expression of genes related to misfolded proteins; 2) (p)ppGpp inhibits DNA 514

primase thus the replication of the chromosome; 3) (p)ppGpp also inhibits 515

transcription of rRNA, affecting the general translation; 4) (p)ppGpp also deregulates 516

LpxC, an enzyme catalyzing the first step of LPS formation; 5) (p)ppGpp binds to RNAP 517

(RNA polymerase) regulating the transcription of many genes; 6) according to certain 518

models, HipA toxin would phosphorylate glutamyl-tRNA-synthetase (GltX), inactivating 519

it and therefore impairing aminoacylation. Empty tRNAs then trigger the stringent 520

response: RelA associates to ribosome and synthesizes (p)ppGpp from GTP/GDP + ATP; 521

7) (p)ppGpp can directly inhibit negative supercoiling of DNA in E. coli, associated with 522

resistance to quinolones; 8) (p)ppGpp induces the transcription of the ribosome 523

modulating factor (Rmf) and hibernation promoting factor (Hpf), which play a role in 524

ribosome dimerization, typical from persister cells. (p)ppGpp is also involved in 525

immune evasion, virulence and human pathogenesis. 526

Figure 2. A. Molecular mechanisms underlying bacterial persistence. B. Different models 527

explaining the involvement of (p)ppGpp in persistence and representative 528

publications. 529

Table 1. Summary of the main ppGpp models. 530

Table 2. Summary of the main treatments having ppGpp as a target. 531

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Acknowledgements 541

This study was funded by grant PI16/01163 and PI19/00878 awarded to M. Tomás within the 542

State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological 543

Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General 544

Directorate for Evaluation and Promotion of Research - European Regional Development Fund 545

"A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the 546

Research in Infectious Diseases (REIPI, RD16/0016/0006 and RD16/0016/0011) and by the 547

Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, 548

http://www.seimc.org/). M.Tomás was financially supported by the Miguel Servet Research 549

Programme (SERGAS and ISCIII). Fernández-García postdoctoral fellowship from the 550

Diputation A Coruña (Xunta Galicia). TJK is supported by a National Health and Medical 551

Research Council Early Career Fellowship (GNT1088448). 552

AUTHOR CONTRIBUTIONS 553

O.P., L.B., I.B., L.F-G., A.A., M.L., wrote the manuscript; G.B., R.C., R.G-C., T.K.W., M.T., 554

supervised and revised the manuscript. 555

TRANSPARENCY DECLARATIONS 556

All authors declare no conflict of interest 557

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856

857

858

Table 1. 859

Rmf: ribosome modulation factor, Hpf: hibernation promoting factor. *Some of these publications have been retracted due to the contamination with the 860

Φ80 bacteriophage causing artefacts in the results 861

Year Journal Author Model References

2014 Cell Maisonneuve E. and Gerdes

K.

(p)ppGpp induces persistence by activating TA loci via

PolyP and Lon protease in E. coli K-12 strain MG1655.

(42, 71-73)*

(3, 65, 109)

2016 Scientific Reports Chowdhury N., Kwan B.W,

and Wood T.K.

The formation of persister cells in E. coli K-12 strain

MG1655 is attributed to production of any toxic protein

(e.g., MazF, RelB and YafO) and (p)ppGpp is not

essential but increases persistence by 1000X.

(75)

2019 Science Signalling Pontes M.H. and Groismann

E.A.

Low cytoplasmatic Mg2+ induces S. typhimurium

tolerance to antibiotic independently of (p)ppGpp and

TA modules. However, (p)ppGpp reduces antibiotic

tolerance under certain conditions.

(49)

2020 Biochemical and

Biophysical Research

Communication

Song S. and Wood T.K. (p)ppGpp generates persister cells directly by

inactivation of ribosomes via Rmf and Hpf.

(83)

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862

Table 2. 863

*Andresen and colleagues rejected this hypothesis two years later, questioning its specificity for (p)ppGpp and for biofilm-forming cells (96). 864

865

866

867

Year Author Strategy Mechanism of action References

2012 Wexselblatt and

colleagues

(p)ppGpp analogs inhibit Rel

protein: relacin

Relacin produced death of B. subtilis with an IC50=200

μM, prevention of sporulation and biofilm formation

and induction of a prolonged exponential phase.

(91)

2012 De la Fuente-Núñez and

colleagues

Inhibition of (p)ppGpp

accumulation: 1037 peptide

1037 reduced expression of flagella-associated genes

that favorize biofilm establishment in P. aeruginosa and

B. cenocepacia. It also reduced the swarming motility.

(95)

2014 De la Fuente-Núñez and

colleagues

Inhibition of (p)ppGpp

accumulation: 1018 peptide

1018 marked (p)ppGpp for degradation*: broad anti-

biofilm activity against Gram positive and negative

bacteria and lack of effect for planktonic cultures.

(94)

2017 Syal and colleagues (p)ppGpp analog to inhibit

Rel protein: modification of

relacin

Impairing of biofilm formation by M. smegmatis and

arising of elongated cells. Lack of toxicity, good

permeability to human lung epithelial cells.

(92)

2019 Dutta and colleagues (p)ppGpp analog to inhibit

Rel protein: compound X9

Highest inhibitory activity against Rel protein: IC50 of

∼15 μM against purified Rel of M. tuberculosis.

Enhancement of susceptibility against isoniazid.

(93)

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